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We previously described an adult-onset distal myopathy (MPD3, OMIM # 610099) in a large Finnish family with a dominant mode of inheritance. Small hand muscles (intrinsic, thenar and hypothenar) were first involved with spread to the lower legs and later proximal muscles. Dystrophic changes with rimmed vacuoles and cytoplasmic inclusions were observed in muscle biopsies at advanced stage. The causative variant was identified in the HNRNPA1 gene. This was a heterozygous deletion of the DNA segment chr12:54677979-54678138 spanning the second last exon 10 of the gene. Whole RNA sequencing showed that the main RNA product of the mutant allele results from splicing of exon 9 to 11 and encodes the predicted protein p.Gly356Asnfs*4. Additional likely disease-causing HNRNPA1 variants were identified in other families with compatible phenotypes. Functional characterization of p.Gly356Asnfs*4 and other novel variants in cell culture models is ongoing to characterize their effects on protein properties and on stress granule dynamics. [ABSTRACT FROM AUTHOR]

Muelas N, Hackman P, Luque H, Suominen T, Espinós C, Garcés-Sánchez M, Sevilla T, Azorín I, Millán JM, Udd B, Vílchez JJ. Spanish MYH7 founder mutation of Italian ancestry causing a large cluster of Laing myopathy patients. Laing myopathy is a distal myopathy caused by mutations in the tail of the slow beta-myosin heavy chain gene MYH7. A large cluster of patients belonging to different families, with Laing myopathy due to p.K1729del mutation, was found in the Safor region, Spain. The same mutation was previously reported in an American family with Italian ancestry. The possibility that p.K1729del in MYH7 might be a founder mutation in the Safor patients and the chance of a common origin with the Italian-American family mutation was investigated by haplotype analyses, mutation data origin estimation and historical inquiry. Our results show that the p.K1729del in MYH7 harboured by patients from the Safor indeed is a founder mutation. A common ancestral origin of this mutation in the Spanish and Italian families is also suggested because they all share a core SNP haplotype at locus MYH7. Data estimation yields the origin of the mutation in the Safor at the beginning of the XVII century, when the Moorish were spelt and the region was resettled with Italian families. [ABSTRACT FROM AUTHOR]

Tibial muscular dystrophy (TMD) is caused by mutations in the TTN gene. TTN is transcribing a >100 kb long mRNA, coding for the sarcomeric, largest known human protein, titin, spanning one half sarcomere in striated muscle. Titin (TTN) binds many other proteins and has important structural, mechanical and regulatory roles in muscles. The first TMD causing mutation, FINmaj a deletion/insertion of eleven base pairs, in the last 363:rd exon, Mex6, was reported by our group in 2002. Several different TTN mutations in the last two exons causing TMD have since been reported in European families. Due to its giant size and complexity analysis of TTN by conventional Sanger sequencing has been difficult and laborous. Usually only certain selected parts of the gene has been screened. Therefore the general spectrum and incidence of titinopathies has been underestimated. Next generation sequencing (NGS) methods have enabled an extensive increase in identification of mutations, and more than 120 TTN mutations have been reported in patients with at least 10 different conditions. In addition to using exome sequencing we have developed a targeted NGS custom panel, MyoCap, with the coding exons and UTRs of 236 muscular dystrophy related genes, including all exons of TTN. Due to the giant size of the gene, rare and novel TTN variants are now identified in many individuals analysed. Thus we have found more than ten patients with previously un-described phenotypes, with potential disease causing variants. This puts challenges in assessing the relevance and real correlation of these mutations with a certain disease phenotype. Since TTN is very repetitive, some regions are not well covered by NGS techniques. Insertion/deletion mutations may be missed in the variant calling. All this adds challenges to the development of the NGS methods and analysis of the results. Moreover, usage of the NGS for TTN requires large repertoire of RNA and protein techniques for the assessment of new variants. [ABSTRACT FROM AUTHOR]

Welander distal myopathy (WDM) is a late onset disease caused by a mutation in the C-terminal region of the TIA1 gene. The secondary molecular events resulting from the causative TIA1 mutations to the preterm death of muscle cells in WDM are mostly unknown. In order to identify downstream pathogenetic mechanisms we explored WDM biopsies genetic profile by expression profiling. We compared the changes to the expression profile of the phenotypically overlapping tibial muscular dystrophy (TMD). Six WDM patient biopsies and five healthy control muscle biopsies were used and the expression data was analyzed using Ingenuity Pathway Analysis. We identified biochemical and genetic pathway changes distinctive to WDM, such as 14-3-3 mediated apoptosis, cleavage and polyadenylation of pre-mRNA, protein trafficking and transport, and oxidative phosphorylation pathway changes. We also identified shared changes with TMD that result in the similarity seen in both these distal myopathies, such as SAP-JNK apoptosis, P70S6 mTOR signalling, mitochondrial dysfunction, and protein ubiquitination pathway changes. TIA1 is a key component of stress granules, a mechanism used to protect other cellular mechanisms during stress. The unique oxidative phosphorylation changes we identified by expression profiling may be associated with increased oxidative stress in WDM. TIA1 is also involved in polyadenylation and splicing of mRNA and the identified changes in these pathways in our study suggest mutated TIA1 affects these pathways in WDM. In addition, both WDM and TMD have common pathological changes in P70S6-mTOR signalling, SAP-JNK apoptosis and protein ubiquitination signalling pathways, which have been reported in other rimmed vacuolar myopathies. [Copyright &y& Elsevier]

Abstract: Welander distal myopathy (WDM) is a late onset, autosomal dominant distal myopathy prevalent in Finland and Sweden. First symptoms of finger extensor weakness occur after age 40, with progression to all hand muscles and ankle dorsiflexion. Rare homozygotes show also proximal muscular weakness, earlier onset and faster progression. WDM is linked to chromosome 2p13 with a maximal linked region of interest of <800kb. Initially all genes were sequenced, later the whole linked genomic region was sequenced with targeted high throughput- and Sanger sequencing, and all known transcripts from the region were sequenced on the cDNA level. Finally a presumably causative gene defect, was revealed in a linked gene. The mutation was not seen in 202 normal Finnish control samples, but was present in all 56 analysed WDM samples previously known to carry the linked haplotype. The gene is coding for an RNA binding protein, that has not been reported mutated in any neuromuscular or other disorders. Immunohistochemical analysis showed predominantly nuclear localization of the protein with some cytoplasmic labelling. Western blotting showed a weaker band in muscle tissue from a homozygote WDM patient indicating either a change of protein tertiary structure with decreased antibody affinity or a decreased amount of total protein. The known functions of the gene are related to cell responses in stress suggest that understanding its malfunctions in muscle disease may have implications for many other degenerative disorders. [Copyright &y& Elsevier]

Distal myopathies are a group of rare progressive genetic muscle disorders typically causing weakness and atrophy in the feet and/or hand muscles. The family under study is affected by an adult-onset dominant distal myopathy and the objective was to identify the pathogenic variant causing the disease. After negative gene panel and exome data, linkage analysis was conducted with genotyped single nucleotide polymorphisms to identify loci that co-segregated with the symptoms. Rare small-scale variants were analyzed with short-read whole exome and genome sequencing. Structural variants (SV) and repeat expansions were detected from whole genome sequenced samples with bioinformatic tools. SVs were further revealed with optical genome mapping. A muscle biopsy of a patient was used for untargeted proteomic profiling to recognize dysregulated proteins, and RNA sequencing was performed to identify splicing alterations and altered transcriptional regulation. Genetic regions identified by linkage analysis did not contain rare segregating variants likely to be pathogenic. Linkage analysis was then repeated several times with different parameters, to account for possible presymptomatic carriers, and a patient with uncertain disease status. But none of the potential new linked regions did contain any potentially promising variants. Proteomic profiling identified over 200 dysregulated proteins, many of which were encoded by muscle-associated genes but were not located in any of the linked regions and did not contain rare segregating variants. RNA sequencing detected splicing alterations in five genes and expression changes in two genes, which were all seemingly unrelated to muscle disorders. To date we haven't been able to identify the causative variant, but further analyses are ongoing. The cause may turn out to be a complex mechanisms such as digenic/oligenic inheritance or limitations of the current next-generation sequencing. [ABSTRACT FROM AUTHOR]

With 121 exons, OBSCN codes for one of the largest mRNAs in the human genome. It is highly expressed in skeletal muscles. It is prone to extensive alternative splicing, several of which produce mRNAs that code for a family of proteins known as "obscurins". Obscurin is associated with neuromuscular functions such as myofibrillogenesis, hypertrophic response and cytoskeletal arrangements. It is known to interact with the giant sarcomere protein titin. Furthermore, it is associated with a number of cardiac and skeletal muscle diseases. In this study, we aim to discover a detailed map of splicing and isoform expression of OBSCN in human skeletal and cardiac muscle development. Therefore, we analyze pre- and post-natal skeletal and cardiac muscle-RNAseq data from 75 individuals (i.e. 45 adult muscles, 7 adult hearts, 20 fetal muscles and 3 fetal hearts). We apply IntEREst R/Biocondcutor to analyze exon inclusion (or skipping), together with other alternative splicing events (e.g. alternative 3'/5' splicing, and alternative first/last exon inclusion) in OBSCN, across skeletal/cardiac muscle development. We use PSIVIS to visualize the Percentage Spliced In (i.e. PSI) for the inclusion of OBSCN exons in the studied sample groups. Our findings improve the clinical and prognostic interpretation of OBSCN exonic variants and their effects in different stages of human life. An interactive visualization of the OBSCN exon inclusion values is available at https://gacatag.shinyapps.io/OBSCN_PSIVIS/. [ABSTRACT FROM AUTHOR]

The molecular diagnosis of muscle diseases poses challenges due to phenotypic variability and genetic heterogeneity. Despite advances in sequencing, many cases remain unsolved, prompting re-analysis with the hope that recent findings would resolve previously ambiguous variants and genes. This happens especially in the case of singletons with no segregation data to assist in the variant interpretation. Sequencing singletons has led to larger cohorts of unsolved cases, which are manually laborious to re-analyse. Standardisation and automation can streamline analysis and re-analysis. Bioinformatic tools such as the gene prioritisation tool Exomiser leverage structured clinical annotations (Human phenotype ontologies) and variant in silico predictions to rank genes and variants, reducing potential ones to a handful, comparing phenotypes to animal models and protein-protein interactions. It can also prioritise phenotypically relevant genes not yet associated with the disease. By combining unsolved singletons to a sample cohort with over 250 samples, we gain essential data to rule out pathogenically called sequencing artefacts by recognising internally reoccurring variants. A cohort approach also supports novel findings if the same gene is seen as pathogenic in several samples. This approach has the potential to uncover previously elusive pathogenic variants and, novel disease genes. Currently utilised with WES with potential to expand to WGS to discover even variants beyond the coding regions. To date, this re-analysis approach has resulted in 10 samples with variants previously missed in known disease genes, and five samples with variants in novel disease genes. By expanding the cohort and periodically re-analysing the cohort automatically with Exomiser we expect to clarify even more cases as databases get updated with new research results. [ABSTRACT FROM AUTHOR]

Calsequestrin-1 (CASQ1) is a calcium-binding protein with high expression in fast twitch fibers of skeletal muscle. Due to its high binding capacity and low affinity for calcium, CASQ1 is the most crucial Ca2+ buffering protein in the sarcoplasmic reticulum, facilitating rapid Ca2+ release to initiate muscle contraction. CASQ1 polymerizes upon Ca2+ binding, which in turn increases its ability to bind Ca2+. The C-terminal CAS domain mediates polymerization as well as interactions with other sarcoplasmic reticulum proteins. Variants in CASQ1 have been associated with vacuolar myopathy with CASQ1 aggregates. Six pathogenic variants have been published to date. The disease is an adult-onset, often slowly progressive muscle disorder, the typical symptoms being proximal and/or distal muscle weakness, myalgia, exercise intolerance, cramps, and fatigue. We have identified three novel dominant CASQ1 variants in seven patients with mild adult-onset myopathy: p.Glu89Lys, p.Ser356Gly, and p.Gly383Alafs*39 (RefSeq NP_001222.3). All variants are predicted to damage the protein. The variant p.Gly383Alafs*39 deletes the aspartic acid residues of the CAS domain and the protein is extended by 24 amino acids. Transfection of CASQ1 -wt and CASQ1 -Ser356Gly to HeLa cells resulted in polymeric CASQ1 networks, whereas CASQ1 -Gly383Alafs*39 ended up in aggregates and CASQ1 -Glu89Lys appeared monomeric. Treatment with the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) inhibitor thapsigargin revealed that CASQ1-Gly383Alafs*39 and CASQ1 -Glu89Lys remain aggregated or monomeric, respectively, also when the ER is depleted of Ca2+. Experiments measuring the Ca2+ binding ability of the variants are underway. In conclusion, this study adds to our understanding of CASQ1-related myopathies. [ABSTRACT FROM AUTHOR]

The TTN gene is composed of 364 exons (363 coding) and encodes for titin, the largest protein in nature. Pathogenic TTN variants can result in a wide spectrum of skeletal muscle and cardiac disorders known as Titinopathies. These differ in inheritance patterns, onset age, disease course and severity. Due to the vast heterogeneity of genetic variants causing TTN -affected skeletal disorders, understanding genotype-phenotype correlations in patients remain challenging. The biological mechanisms underlying disease-causing variants specific to Titinopathy patients are still elusive. Investigating key driver genetic backgrounds causing the biological pathomechanisms is crucial for developing effective therapeutic strategies. RNA-sequencing emerges as a valuable technique for analysing transcriptomic data and exploring gene expression profiles of patient and control samples. To elucidate common pathomechanisms in Titinopathies, including dominant FINmaj mutated Tibial Muscular Dystrophy (TMD) and biallelic recessive Titinopathies, we conducted an extensive differential gene expression (DGE) analysis using three RNA cohorts from human muscle biopsies: two polyA enriched and one rRNA depleted batch corrected cohort. Human DGE analysis, performed with DESeq2, identified 265 commonly upregulated genes and 147 commonly downregulated genes in the Titinopathy cohorts. To validate and understand the biological significance of these findings, we incorporated data from a mouse model with homozygous Ttn FINmaj variants. MATN2, COL5A1, LUM, COL3A1, MFAP5 and ASPN genes are commonly upregulated in both human and mouse cohorts accounting for the structural integrity of the extracellular matrix. YBX3 is a commonly downregulated RNA-binding protein known to take part in skeletal muscle proliferation and differentiation. These results highlight molecular compensatory mechanisms for structural muscle impairment and cellular stress response. Further complementation with proteomics analysis will provide more and comprehensive insights on the biological mechanisms of titinopathies. [ABSTRACT FROM AUTHOR]

Background and purpose: The aim was to determine the genetic background of unknown muscular dystrophy in five French families. Methods: Twelve patients with limb girdle muscular dystrophy or distal myopathy were clinically evaluated. Gene mutations were identified using targeted exon sequencing and mutated DNAJB6 was tested in vitro. Results: Five patients presented with distal lower limb weakness whilst others had proximal presentation with a variable rate of progression starting at the mean age of 38.5 years. Two novel mutations (c.284A>T, p.Asn95Ile, two families; and c.293_295delATG, p.Asp98del, one family) as well as the previously reported c.279C>G (p.Phe93Leu, two families) mutation in DNAJB6 were identified. All showed a reduced capacity to prevent protein aggregation. Conclusions: The mutational and phenotypical spectrum of DNAJB6-caused muscle disease is larger than previously reported, including also dysphagia. The originally reported c.279C>G (p.Phe93Leu) mutation is now identified in four different populations and appears to be a mutational hotspot. Our report confirms that some DNAJB6 mutations cause distal-onset myopathy and hence DNAJB6 defects should be considered broadly in dominant muscular dystrophy families. [ABSTRACT FROM AUTHOR]

Background and purpose The aim was to identify potential genetic risk factors associated with sporadic inclusion body myositis ( sIBM). Methods An association based case−control approach was utilized on whole exome sequencing data of 30 Finnish sIBM patients and a control cohort ( n = 193). A separate Italian cohort of sIBM patients ( n = 12) was used for evaluation of the results. Results Seven single nucleotide polymorphisms were identified in five genes that have a considerably higher observed frequency in Finnish sIBM patients compared to the control population, and the previous association of the genetic human leukocyte antigen region was confirmed. Conclusions All seven identified variants could individually or in combination increase the susceptibility for sIBM. [ABSTRACT FROM AUTHOR]

ACTN2 encodes alpha-actinin-2, a protein expressed in human cardiac and skeletal muscle. The protein is located in the sarcomere Z-disk, and functions as a link between the anti-parallel actin filaments. Further, alpha-actinin-2 contributes to sarcomere structure and stability by binding N-terminal titins. Previously, ACTN2 mutations have been associated with cardiomyopathy, without involvement of skeletal muscle. Recently, however, ACTN2 mutations have been reported to show association with novel congenital and distal myopathies. These previously reported variants are spread across the gene in varying locations, but the potential clustering effect of pathogenic locations is poorly understood. Similarly, the genotype-phenotype correlations of these variants are not well defined. Here we review the previously reported ACTN2 -related molecular and clinical findings and present an additional variant, c.1840-2A>T, that further expands the mutation and phenotypic spectrum of ACTN2 -related conditions. We found eight unique ACTN2 variants associated with a skeletal muscle disease, and 16 variants with cardiac disease. Our results show increasingly clear genetic and functional evidence that underscores the central role of alpha-actinin-2 in the muscle tissue and myopathy. Still, limited segregation and functional data are available to support the pathogenicity of most previously reported missense variants. Thus, clear-cut genotype-phenotype correlations are currently only demonstrated for some ACTN2 -related myopathies. [ABSTRACT FROM AUTHOR]

Tibial muscular dystrophy (TMD) is a dominant, late-onset distal titinopathy. It was first described in Finnish patients three decades ago and the presence of a founder mutation has resulted in a prevalence of TMD in over 1,000 patients in Finland. Nevertheless, no natural history studies have previously been carried out for TMD or any other titinopathies. For the study, we were able to obtain longitudinal follow-up data of the evolution of clinical muscle weakness over 15 years in 137 Finnish TMD patients. Our study suggests that the first symptoms of mild walking difficulties due to the muscle weakness in distal lower limbs occasionally can occur already in early adulthood (16-35 years). Because the disease has a mild onset and progression, patients tend to recognize signs of disease only after a longer period of early symptoms if they are not aware of having a disease from the family history. In the early stages of the disease muscle weakness occurred preferentially in the anterior compartment muscles of lower limbs resulting in walking difficulties such as slabbing with the feet and tripping. In later stages, proximal lower limb and also upper limb weakness occurred but remained moderate even at very high ages. At last examination, the use of walking aids was common after 80 years of age, but full wheelchair dependency did not develop. [ABSTRACT FROM AUTHOR]

Pathogenic mutations in the low complexity domain of many RNA binding proteins have been reported in both neurogenic and myopathic disorders. Frequent involvement of multiple organs in patients often leads to associated diseases classified as multisystem proteinopathies (MSP) because of the shared aggregate pathology in the affected tissues. These mutations and associated molecular pathomechanisms are part of the dysfunctional cellular clearance of protein aggregates and stress responses. Interestingly, the same causative variant has often been observed in patients with ALS and large families with primary myopathies with or without ALS. Recently, we identified the c.118G>T; p.D40Y variant in ANXA11 (NM_0145869.1) segregating with an autosomal dominant myopathy phenotype in five large Greek families from the relatively isolated island of Aegean Sea. However, this variant has been reported in French ALS patients and Brazilian MSP families with ALS patients. In our cohort, none of the patients have an ALS phenotype or family history of ALS. The age of onset in our patients was in the 3rd-5th decade of life. Muscle MRI shows myopathic fatty degeneration-replacement of adductors, semitendinosus, semimembranosus, and gastrocnemius muscles. However, none of our patients report any axial involvement with head drop described in the Brazilian MSP families with the same mutation. We present strong genetic evidence that ANXA11 p.D40Y causes the observed phenotype in the Greek families. Further studies will unravel the additional molecular factors that can explain the involvement of different target organs causing either ALS or the primary myopathy phenotype. [ABSTRACT FROM AUTHOR]

Myopathies are a clinically and genetically heterogeneous group of disorders that cause progressive weakness and atrophy of muscles. Mutations in more than 200 different genes are known to cause many of these disorders and in less obvious disorders several genes may have to be sequenced in order to identify the correct diagnosis in a patient. The large number of possible candidate genes, overlapping phenotypes as well as an enormous size of some of the genes e.g. DMD , TTN and NEB constitute difficult challenges for clinical diagnostics. Molecular characterization is nevertheless important for the final diagnosis and accurate management of the diseases. Targeted next-generation sequencing (NGS) is an efficient and cost-effective method to sequence several genes simultaneously. A targeted NGS custom panel with a total size of 1.3 Mb was designed for the coding exons and UTRs of 180 myopathy related genes. For DNA capture a custom NimbleGen SeqCap EZ Choice Library was designed. Sequencing was performed at The Institute for Molecular Medicine Finland (FIMM) using Illumina HiSeq 1500 with sequencing depth of 100×, and files were processed with their Variant Calling Pipeline. In this part of the study DNA samples of 67 patients negative for previous candidate gene approaches were sequenced. According to preliminary results definite diagnosis based on disease-causing mutations was obtained directly in 15 patients and probable disease-causing mutations were found in another 12 patients. Ten patients had potential disease-causing TTN mutations with previously undescribed phenotypes and the in depth study of the results is still ongoing. [ABSTRACT FROM AUTHOR]

Abstract: Nemaline myopathy (NM) constitutes a heterogeneous group of congenital myopathies. Mutations in the nebulin gene (NEB) are the main cause of recessively inherited NM. NEB is one of the most largest genes in human. To date, 68 NEB mutations, mainly small deletions or point mutations have been published. The only large mutation characterized is the 2.5kb deletion of exon 55 in the Ashkenazi Jewish population. To investigate any copy number variations in this enormous gene, we designed a novel custom comparative genomic hybridization microarray, NM-CGH, targeted towards the seven known genes causative for NM. During the validation of the NM-CGH array we identified two novel deletions in two different families. The first is the largest deletion characterized in NEB to date, (∼53kb) encompassing 24 exons. The second deletion (1kb) covers two exons. In both families, the copy number change was the second mutation to be characterized and shown to have been inherited from one of the healthy carrier parents. In addition to these novel mutations, copy number variation was identified in four samples in three families in the triplicate region of NEB. We conclude that this method appears promising for the detection of copy number variations in NEB. [Copyright &y& Elsevier]

Mahjneh I, Lamminen AE, Udd B, Paetau AE, Hackman P, Korhola OA, Somer HVK. Muscle magnetic resonance imaging shows distinct diagnostic patterns in Welander and tibial muscular dystrophy. Acta Neurol Scand 2004 DOI: 10.1111/j.1600-0404.2004.00283.x © Blackwell Munksgaard 2004. This is a report on a retrospective muscle magnetic resonance imaging (MRI) study on 11 patients affected by Welander distal myopathy (WDM) and 22 patients with tibial muscular dystrophy (TMD) carried out in order to define the pattern and characteristics of muscle involvement. WDM patients showed involvement of gastrocnemius, soleus, tibial anterior (TA) and extensor digitorum longus (EDL), as well as hamstrings and hip adductor muscles. TMD patients showed involvement of the TA and EDL muscles, and in some patients also hamstring and posterior compartment muscles of the legs. Some patients showed asymmetry of muscle involvement. We conclude that muscle MRI examination proved to be very useful in the determination of the exact pattern of muscle involvement in WDM and TMD. Clinical testing using the Medical Research Council scale is not sensitive enough to establish the pattern of muscle involvement in focal muscle diseases. [ABSTRACT FROM AUTHOR]

Myotonic dystrophy types 1 and 2 are autosomal dominant, multisystemic disorders with many similarities in their clinical manifestations. Myotonic dystrophy type 1 is caused by a (CTG)n expansion in the 3′ untranslated region of the DMPK gene in 19q13.3 and myotonic dystrophy type 2 by a (CCTG)n expansion in intron 1 of ZNF9 in 3q21.3. However, the clinical diagnosis of myotonic dystrophy type 2 is more complex than that of myotonic dystrophy type 1, and conventional molecular genetic methods used for diagnosing myotonic dystrophy type 1 are insufficient for myotonic dystrophy type 2. Herein we describe two in situ hybridization protocols for the myotonic dystrophy type 2 mutation detection. Chromogenic in situ hybridization was used to detect both the genomic expansion and the mutant transcripts in muscle biopsy sections. Chromogenic in situ hybridization can be used in routine myotonic dystrophy type 2 diagnostics. Fluorescence in situ hybridization on extended DNA fibers was used to directly visualize the myotonic dystrophy type 2 mutation and to estimate the repeat expansion sizes. [Copyright &y& Elsevier]

The sudden infant death syndrome has multiple etiologies. Some congenital myasthenic syndromes can cause sudden infant death syndrome by apnea, but the frequency of this etiology is unknown. We report here a young patient with sudden respiratory crises culminating in apnea followed by recovery, against a background of no or variable myasthenic symptoms without dyspnea. One sib without myasthenic symptoms and one sib who only had mild ptosis died previously during febrile episodes. Studies reported by us elsewhere traced the proband''s illness to mutations in choline acetyltransferase. Here, we describe in detail the morphologic investigations and electrophysiologic findings, which point to a presynaptic defect in acetylcholine resynthesis or vesicular filling, in the proband. Analysis of DNA from a sib who previously died of sudden infant death syndrome revealed the same choline acetyltransferase mutation. Thus, mutations in choline acetyltransferase may be a cause of sudden infant death syndrome as, theoretically, could other presynaptic myasthenic disorders. [Copyright &y& Elsevier]

Hereditary myopathy with early respiratory failure (HMERF) is an early adult-onset muscular dystrophy, with weakness predominantly in proximal and respiratory muscles. A mutation in the titin kinase domain was originally reported as one cause of the disease. However, the genetic background of HMERF has recently turned out to be more complex, with titin A150/FN3 119 domain mutations playing a central role. The HMERF muscle histopathology consists of myopathic-dystrophic changes with protein aggregation pathology, including autophagic rimmed vacuoles and myofibrillar pathology. A distinctive feature is the presence of cytoplasmic bodies, which are dense, often spherical bodies located either in the fiber periphery or in central regions. Muscle biopsies from HMERF patients with different mutation combinations, including the recessive p.P30091L, the dominant p.C30071R and the kinase domain recessive p.R32450W, were analyzed using immunofluorescence. Various kinds of protein accumulation were observed. The cytoplasmic bodies were positive F-actin (phalloidin), myotilin, ubiquitin, MURF1, DNAJB6 and NBR1, but negative for alphaB-crystallin, desmin, p62 and several titin antibodies, including A-band TTN. Outside the bodies, p62 and NBR1 formed distinct dense aggregates, whereas alphaB-crystallin, desmin and TTN accumulated in more diffuse manner, both in the periphery and central fiber areas. The function or binding partners of the titin FN3 119 domain are not yet known. However, mutations in it trigger a molecular cascade leading to accumulation of ubiquitinated proteins, which exhausts the degradation capacity of the muscle fibers. This finding emphasizes the role of titin as a central signaling protein, regulating protein turnover, proteasomal and autophagic degradation. Both the FN3 119 domain and the kinase domain seem to be key players in this regulation. [ABSTRACT FROM AUTHOR]

Several patients with previously reported titin gene (TTN) mutations causing tibial muscular dystrophy (TMD) have more complex, severe or unusual phenotypes. This study aimed to clarify the molecular cause of these variant phenotypes in proband patients of six European families. Clinical, histopathological and muscle imaging data of patients and family members was reanalyzed and muscle biopsies of the patients were studied by titin Western blotting and RT-PCR. Western blotting showed a more pronounced C-terminal titin abnormality than expected for heterozygous mutants in all six probands, suggesting the existence of additional TTN mutations. RT-PCR indicated unequal mRNA expression of the two TTN alleles in biopsies of four patients, two of them with an LGMD2J phenotype. TTN was analyzed by Sanger sequencing from genomic DNA of the patients. Three patients proved to be compound heterozygotes with novel TTN frameshift mutations combined with previously reported TMD mutations. One LGMD2J patient was heterozygous for the FINmaj TMD mutation with a likely second yet undiscovered mutation. The unequal expression levels of TTN transcripts in these four probands suggested that the expression of the frameshifted allele was severely reduced, probably through nonsense-mediated decay, explaining the observed more severe phenotypes. One Portuguese patient was homozygote for a previously known Spanish TMD mutation. This TMD mutation seems to cause a more severe TMD rather than LGMD2J when homozygous. One Finnish patient had FINmaj mutation combined with a novel missense mutation in the A-band region of titin causing a new phenotype completely different from previously described titinopathies. Our results further expand the complexity of muscular dystrophies caused by TTN mutations and suggest that the coexistence of second mutations may constitute a more common general mechanism of phenotype variability in muscular dystrophies. [Copyright &y& Elsevier]

Abstract: Hereditary myopathy with early respiratory failure (HMERF) is characterized by proximal–distal weakness and respiratory insufficiency in ambulant patients. The clinical onset of the disease is variable, ranging from early to late adulthood. Muscle weakness is typical in neck flexors, limb girdle, and in some cases also in distal lower limb muscles. A prominent early finding is respiratory muscle weakness presenting with an acute respiratory failure in many patients. A dominant mutation in the TTN kinase domain leading to HMERF has been described in a few families from Sweden. Very recently, two reports (in press) revealed a novel dominant A-band mutation in titin gene, TTN. This mutation (g.274375T>C; p.Cys30071Arg) was found in eight HMERF patients from three Swedish families and in 31 patients from three families. In our collaborative study we have identified the identical mutation in one Finnish family with four affected HMERF patients. In addition, the mutation was identified in one British patient, in one Italian patient and in one additional Swedish family. The patients had the same phenotype with progressive muscle weakness in the lower limbs and respiratory muscles, cytoplasmic bodies and rimmed vacuolar myopathy in muscle biopsy. Muscle MRI findings are pathognomonic with fatty degenerative changes in semitendinosus, obturatorius, sartorius, gracilis and iliopsoas muscles with variable involvement of anterior and lateral compartment and tibialis posterior on the lower legs. All patients carried the same haplotype including markers D2S2173 and D2S385 at the mutational locus. The shared genomic region is less than 3.3Mb in size suggesting an ancestral founder, and indicating the existence of many undiagnosed families with this mutation. [Copyright &y& Elsevier]